Abstract

Background

   Perioperative chemotherapy is the standard of care for patients with
   locally advanced gastric and gastroesophageal junction cancer. Recent
   evidence demonstrated the addition of programmed cell death protein 1
   (PD-1) inhibitors enhanced therapeutic efficacy. However, the
   mechanisms of response and resistance remain largely undefined. A
   detailed multiomic investigation is essential to elucidate these
   mechanisms.

Methods

   We performed whole-exome sequencing, whole-transcriptome sequencing,
   multiplex immunofluorescence and single-cell RNA sequencing on matched
   pretreatment and post-treatment samples from 30 patients enrolled in an
   investigator-initiated Phase 2 clinical trial ([55]NCT04908566). All
   patients received neoadjuvant PD-1 inhibitors in combination with
   chemotherapy. A major pathologic response (MPR) was defined as the
   presence of no more than 10% residual viable tumor cells following
   treatment.

Results

   Before treatment, the positive ratio of CD3+T cells in both the tumor
   parenchyma and stroma was significantly higher in the non-MPR group
   compared with the MPR group (p=0.042 and p=0.013, respectively). Least
   absolute shrinkage and selection operator regression was employed for
   feature gene selection and 13 genes were ultimately used to construct a
   predictive model for identifying MPR after surgery. The model exhibited
   a perfect area under curve (AUC) of 1.000 (95% CI: 1.000 to 1.000,
   p<0.001). Post-treatment analysis revealed a significant increase in
   CD3+T cells, CD8+T cells and NK cells in the tumor stroma of MPR
   patients. In the tumor parenchyma, aside from a marked increase in
   CD8+T cells and NK cells, a notable reduction in macrophage was also
   observed (all p<0.05). Importantly, forkheadbox protein 3 (FOXP3), the
   principal marker for regulatory T cells (Treg) cells, showed a
   significant decrease during treatment in MPR patients. FOXP3 expression
   in the non-MPR group was significantly higher than in the MPR group
   (p=0.0056) after treatment. Furthermore, single-cell RNA sequencing
   analysis confirmed that nearly all Treg cells were derived from the
   non-MPR group.

Conclusions

   Our study highlights the critical role of dynamic changes within the
   tumor immune microenvironment in predicting the efficacy of neoadjuvant
   combined immunochemotherapy. We examined the disparities between
   MPR/non-MPR groups, shedding light on potential mechanisms of immune
   response and suppression. In addition to bolstering cytotoxic immune
   responses, specifically targeting Treg cells may be crucial for
   enhancing treatment outcomes.

   Keywords: Gastric Cancer, Neoadjuvant, Immunotherapy, Tumor
   microenvironment - TME
     __________________________________________________________________

Key messages.

     * The addition of immunotherapy to perioperative chemotherapy
       enhances therapeutic efficacy, but the mechanisms of response and
       resistance remain unclear. We performed multiomic investigation on
       matched pretreatment and post-treatment tissue samples from 30
       patients with locally advanced gastric and gastroesophageal
       junction cancer. Our study highlights the critical role of dynamic
       changes within the tumor immune microenvironment in predicting the
       efficacy of immunotherapy combined with chemotherapy. In addition
       to bolstering cytotoxic immune responses, specifically targeting
       regulatory T cell cells may be crucial for enhancing treatment
       outcomes.

Introduction

   Gastric and gastroesophageal junction (G/GEJ) cancer is the fifth most
   common cancer and the fourth most lethal cancer worldwide.[56]^1 G/GEJ
   adenocarcinoma accounts for 95% of G/GEJ cancers. For patients with
   locally advanced G/GEJ adenocarcinoma, perioperative chemotherapy
   combined with surgical resection is recommended. This combined
   treatment approach maximizes systemic therapy while reducing the stage
   of locally advanced tumors and improving the eradication of residual
   disease and prevention of metastasis.[57]2,[58]4

   In the FLOT4 study, patients were randomized to receive either the FLOT
   regimen (5-fluorouracil, oxaliplatin, and docetaxel) or the ECF/ECX
   regimen (epirubicin, cisplatin, and 5-fluorouracil/capecitabine) as
   perioperative treatment. The results suggest that the FLOT regimen can
   improve the R0 resection rate (78% vs 85%) and pathological complete
   response rate (pCR, 6% vs 16%). In terms of long-term prognosis, the
   FLOT regimen led to significant improvements in overall survival (OS,
   35 vs 50 months) and disease-free survival (DFS, 18 vs 30
   months).[59]^2 Moreover, the RESOLVE study conducted in China showed
   that the SOX (S1 and oxaliplatin) regimen during the perioperative
   period improved 3-year DFS compared with the XELOX (capecitabine and
   oxaliplatin) regimen as adjuvant therapy (62.02% vs 54.78%).[60]^5
   Therefore, perioperative chemotherapy for G/GEJ adenocarcinoma can
   increase the R0 resection rate, reduce recurrence, and improve OS.
   However, the prognosis of patients with locally advanced G/GEJ cancer
   remains unsatisfactory.

   In the past decade, immune checkpoint inhibitors that target programmed
   cell death 1 (PD-1) have been successful in the treatment of malignant
   tumors. Several Phase 3 clinical studies have confirmed the significant
   efficacy of chemotherapy combined with a PD-1 inhibitor in patients
   with advanced or metastatic G/GEJ cancer, and this regimen is also
   recommended as the standard first-line treatment.[61]6,[62]10 Phase 2
   trials have also reported favorable antitumor activity with PD-1
   inhibitors plus chemotherapy in patients with locally advanced G/GEJ
   cancer.[63]11,[64]14 The interim analysis of KEYNOTE-585, a Phase 3
   trial aiming to explore the antitumor activity of neoadjuvant and
   adjuvant pembrolizumab (a PD-1 inhibitor) plus chemotherapy in patients
   with locally advanced G/GEJ adenocarcinoma, showed that perioperative
   pembrolizumab vs placebo improved the pCR rate (12.9% vs 2.0%,
   p<0.00001).[65]^15 Tumor regression after neoadjuvant therapy was
   significantly correlated with DFS and OS.[66]^16 The results of a study
   of 551 patients with G/GEJ cancer undergoing neoadjuvant treatment
   showed that, compared with patients with a tumor regression grade of 2
   or 3, patients with a major pathological response (MPR) had a
   significant improvement in the 3-year OS rate (70.9% vs 48.8%).[67]^17
   Overall, these studies demonstrated the efficacy of the addition of a
   PD-1 inhibitor for the treatment of locally advanced G/GEJ cancer, but
   the improvement in the pCR rate is very limited. Thus, patients who
   would benefit from PD-1 inhibitors combined with chemotherapy urgently
   need to be identified. However, biomarkers remain unclear and
   controversial. Moreover, the tumor immune microenvironment of locally
   advanced G/GEJ cancer and the effects of PD-1 inhibitors combined with
   chemotherapy are not fully understood. Therefore, comprehensive data
   analysis is needed to determine the underlying reasons and guide
   interventions to improve treatment efficacy.

   In the present study, we integrated multiomics data obtained from
   whole-exome sequencing (WES), whole-transcriptome sequencing (WTS),
   multiplex immunohistochemistry (mIHC) analysis and single-cell RNA
   sequencing. By comparing the genome and tumor microenvironment of MPR
   and non-MPR patients, we screened for effective predictive biomarkers
   of the response to neoadjuvant PD-1 inhibitors combined with
   chemotherapy. Moreover, we characterized the changes in the tumor
   genome and tumor microenvironment in patients before and after
   treatment to elucidate potential mechanisms of response and resistance.

Materials and methods

Data collection

   This study included patients with locally advanced G/GEJ cancer from a
   Phase 2 clinical trial ([68]NCT04908566) who had available and adequate
   primary tumor tissue samples for multiomics molecular analysis before
   and after neoadjuvant therapy. Tumor, node, metastases staging was
   determined by ultrasound gastroscopy and CT scan before treatment. The
   patients received four cycles of SOX combined with a PD-1 inhibitor,
   then underwent radical surgical resection and continued four cycles of
   SOX combined with a PD-1 inhibitor after surgery. The treatment outcome
   was assessed based on the Response Evaluation Criteria in Solid Tumors
   (RECIST) V.1.1 criteria by two independent pathologists. MPR is defined
   as ≤10% viable tumor cells in the resected primary tumor, excluding
   resected lymph nodes.

   We collected tumor biopsy tissues from patients before treatment for
   WES and WTS to profile gene expression. Furthermore, we conducted mIHC
   to analyze the tumor microenvironment. After radical surgical
   resection, surgical tissues were used to assess the MPR status, WTS was
   used to detect gene expression profiles, and mIHC analysis and
   single-cell RNA sequencing were used to analyze the tumor
   microenvironment and cellular heterogeneity ([69]figure 1). The method
   for accessing the datasets is detailed in the Data Availability
   Statement.

Figure 1. The study design. AUC, area under curve; FOXP3, forkheadbox protein
3; MPR, major pathological response; NK, natural killer; PD-1, programmed
cell death 1; Tac, activated T cells; Tcm, central memory T cells; Tem,
effector memory T cells; Tex, exhausted T cells; TMB, tumor mutation burden;
TPM, transcripts per million; Treg, regulatory T cell; Trm, resting memory
cells; UMAP, Uniform Manifold Approximation and Projection.

   [70]Figure 1
   [71]Open in a new tab

WES and data analysis

   Tissue DNA was extracted from formalin-fixed paraffin-embedded (FFPE)
   tumor tissues using the QIAamp DNA FFPE Tissue Kit (Qiagen, Hilden,
   Germany) following the manufacturer’s standard protocol. Fragments
   between 200 and 400 bp from the sheared tissue DNA were purified
   (Agencourt AMPure XP Kit, Beckman Coulter, California, USA), hybridized
   with capture probes baits, selected with magnetic beads, and amplified.
   Targeted sequencing of the whole-exome region was performed using
   capture-based methods.[72]18,[73]20 The quality and the size of the
   fragments were assessed by high sensitivity DNA kit using Bioanalyzer
   2100 (Agilent Technologies, California, USA). Indexed samples were
   sequenced on NextSeq 500 (Illumina, California, USA) with paired-end
   reads and an average sequencing depth of 1,000× for tissue samples.

   Variant calling and annotation were performed using standardized
   pipelines following previously established methods (Burning Rock
   Biotech, Guangzhou, China).[74]^18 Tumor mutation burden (TMB) per
   patient was computed as a ratio between the total number of
   non-synonymous mutations detected and the total coding region size. The
   mutation count included non-synonymous single nucleotide variants
   (SNVs) and indels detected within the coding region and ±2 bp upstream
   or downstream region.

   Copy number variations (CNVs) were analyzed based on the depth of
   coverage data of capture intervals. Coverage data were corrected
   against sequencing bias resulting from guanine cytosine (GC) content
   and probe design. The average coverage of all captured regions was used
   to normalize the coverage of different samples to comparable scales.
   The copy number was calculated based on the ratio between the depth of
   coverage in tumor samples and the average coverage of an adequate
   number (n>50) of samples without CNVs as references per capture